1. Field of the Invention
The present invention relates to the field of immunology generally, and more particularly to a large scale procedure for producing commercial quantities of highly purified type B hepatitis antigen (HB Ag) which may be used in the commercial production of a vaccine against type B hepatitis infections.
2. Description of Prior Observations and Developments
An antigen detected during the incubation period and early clinical course of post-transfusion serum hepatitis is discussed by Prince, Proc. Nat. Acad. Sci. U.S.A. 60:814-821 (1968). This antigen appears to be identical to the so-called Australia antigen (See Prince, Lancet, 2:462-463 (1968); Blumberg, Sutnick and London, J. Am. Med. Assoc. 207:1895-1896 (1969); and Wright, McCollum and Klatskin, Lancet 2:118-121 (1969), and an exchange of reference reagents has established identity between this antigen and the "hepatitis antigen" of Gocke and Kavey, Lancet 1:1055-1059 (1969). The antigen has been described by Prince, Hargrove, Szmuness, Cherubin, Fontana and Jeffries, N. Engl. J. Med. 282:287-291 (1970) as being specific for the virus of serum hepatitis, a virus which appears to be a major cause of sporadic hepatitis in urban adults, regardless of the presence or absence of parenteral exposure to blood or blood products.
The antigen associated with such serum hepatitis infections has previously been called by a variety of names such as Australia antigen, SH antigen, Au/Sh antigen, HAA, etc., and each of these names has had its supporters. However, each also has inherent defects. Geographic names make life difficult for students and physicians and HAA ignores the evident specificity of this antigen for infections with type B hepatitis virus. The term SH, has the unfortunate connotation that this antigen is associated with a virus transmissible only by "serum" or blood products; however, many lines of evidence now indicate clearly that hepatitis B virus (serum hepatitis virus) is also "infectious". Thus, the continued use of the term "serum hepatitis" is likely to result in more confusion than clarification. For this reason a special subcommittee was appointed by the National Research Council of the U.S. National Academy of Sciences to attempt, among other things, to suggest a better terminology. The terminology chosen returns to the classical terms hepatitis type A virus and hepatitis type B virus, which were employed in the 1940's and 50's. The antigen therefore logically becomes hepatitis B antigen (HB Ag) and the antibody directed towards this antigen becomes hepatitis B antibody (HB Ab). Such terminology will be used herein.
In 1964 Blumberg, Bull, N.Y. Acad. Med. 40:377-386 (1964) described the discovery of what then appeared to be another human serum protein polymorphism. An antibody was detected in the serum of a multiply transfused hemophiliac which reacted in the Ouchterlony technique with an antigen which was not .beta.-lipoprotein and which was found to be present in the sera of a proportion of certain foreign populations and in the sera of some patients with leukemia. The antigen was called the Australia antigen since it was initially detected in the serum of an Australian aborigine. Family studies appeared to support the hypothesis that this antigen was a genetically determined isoantigen. The Australia antigen was subsequently shown to be present in about 25% of institutionalized patients with Down's syndrome.
A chance observation by Blumberg provided grounds for a new interpretation of the prior findings discussed above. Serial blood samples were obtained from a child with Down's syndrome. Although the child did not initially have detectable antigen, a subsequent sample gave positive results. Clinical data revealed that at almost the same time the child developed hepatitis. It was then found that the antigen was present in 5 out of 48 sera from patients with viral hepatitis.
These date were compatible with at least three hypotheses: (1) that the antigen was a genetically determined serum isoantigen whose presence correlated with susceptibility to a variety of diseases or disease agents, e.g., leukemia, mongolism, hepatitis (2) that the antigen was a genetically determined isoantigen whose expression depended on the presence of a "derepressing" virus; and (3) that the antigen was specifically associated with a virus causing one or more of these conditions.
At first, the latter hypothesis was considered to be the least likely, since it was not anticipated that a viral antigen would circulate in healthy carriers in quantities sufficient for detection by an insensitive immunodiffusion assay. However, the results of a collaborative study by Prince and Blumberg of the physical characteristics of the antigen supported the third hypothesis since the antigen was found to be associated with a particle that sedimented at the rate of a small virus-like particle.
A specific association between Australia antigen and hepatitis was, however, not made until the composition of the agar employed for the immunodiffusion test had been changed. The precipitation lines were only then sufficiently clear to permit identity testing. The results obtained with the recently developed immunodiffusion system are described by Prince, Proc. Nat. Acad. Sci. 60:814-821 (1968).
Jokelainen, Krohn, Prince, and Finlayson, J. Virol 6:685-689 (1970) investigated the structural aspects of hepatitis B antigen-containing particles with an electron microscope and confirmed the existence of large spherical particles (ca. 43 nm) and smaller (ca. 20 nm) rod- and sphere-shaped particles. The larger particles seem to include outer and inner membranes and a core as seen by positive staining techniques. The outer membranes of the large particles appear to be similar to the 20-nm diameter spheres and rods known to possess the hepatitis B antigen. Each of the three kinds of particles appears to contain the hepatitis B antigen, because they are all clumped by hepatitis B antiserum.
Occasional large particles have projections with a structure identical to that of the rod forms, and constrictions along these in some instances give rise to an appearance suggesting a series of small spherical particles. These findings appear to agree with the findings of Dane, Cameron and Briggs, Lancet 1:695-698 (1970) that all of these forms are produced de novo and that the small spheres and rods may represent an excessive production of membrane material.
Studies with negative staining have confirmed the virus-like appearance of the large spherical particles. Positive-staining studies have indicated three further points: (i) the larger particles have a double membrane structure; (ii) the central core of the nucleoid-like component contains material which stains with uranyl acetate; and (iii) the small spheres and rods do not contain any core material. Although uranyl acetate cannot be considered a specific stain for nucleoprotein, it is recognized as being taken up preferentially by that material and this would be expected to occur within the core of a virus. Therefore, these findings support the hypothesis that, of the three hepatitis B antigen-containing particles described, the larger 40 to 45 nm particles are most probably the actual hepatitis B virus.
Krugman et al., J. Am. Med. Assoc. 217:41 (1971), have provided preliminary data suggesting that serum containing HB Ag which has been inactivated by boiling for 1 minute is noninfectious, immunogenic and protective when administered to a small group of volunteer children.
Prince, Szmuness, Hargrove, Jeffries, Cherubin and Kellner have presented a comprehensive report on the status of research activities directed to investigating the hepatitis B virus specific antigen in Perspectives in Virology, Vol. 7, Chap. 14, pp. 241-296 (Academic Press, Inc., 1971).
It has thus been shown that HB Ag is present in serum during the incubation period of classical post-transfusion serum hepatitis. Antibody to this antigen has been found in patients who have been multiply transfused, such as patients with hemophilia and Cooley's anemia. The antibody is usually not detected in sera from convalescent patients with typical cases of viral hepatitis. The hepatitis B antigen has been found to be identical with the previously described Australia antigen, and with the "hepatitis antigen" of Gocke. The antigen has been shown to be associated with virus-like particles 20 to 25 nanometers in diameter and has an aqueous density of 1.17 in sucrose. Sera from patients with acute viral hepatitis have been tested for the presence of HB Ag to determine whether this would permit distinction between the two major types of viral hepatitis. None of 4 cases of short incubation MS-1 infection tested had detectable antigen; whereas the antigen was identified in all 8 cases of long incubation MS-2 infection tested. Correspondingly, only one out of 74 cases associated with four epidemics of infectious hepatitis, and none of 19 sporadic cases occurring in children under the age of 14 showed presence of detectable antigen; while 76 of 116 cases (66%) which occurred following exposure to contaminated needles and 25 of 43 post-transfusion cases (58%) were positive.
HB Ag has also been detected in 71 of 129 patients (55%) with viral hepatitis who gave no history of parenteral exposure.
These findings suggest that hepatitis B virus is the major cause of sporadic hepatitis in urban adults regardless of the presence or absence of parenteral exposure to blood or blood products.
HB Ag has been found to be 10 to 100 times more prevalent in tropical populations than in volunteer blood donors in New York City. These findings confirm previous results obtained by testing for the Australia antigen.
Although about 90 to 95% of patients with acute serum hepatitis in whom antigen is detected have detectable HB Ag in the blood only for short periods, some persons develop long lasting hepatitis B antigenemia. Long term persistence of antigen is also seen in clinically well individuals in all populations which have been examined.
Two to 3 percent of drug users without evidence of acute hepatitis have detectable quantities of HB Ag. By comparison, the antigen can be detected in only 0.1 percent of the volunteer blood donors in New York City. Paid blood donors, who have been reported to be at least 10 times as likely as volunteer donors to transmit hepatitis, have also been found to be 12.5 times as likely as the volunteer donors to show the presence of HB Ag in their blood.
Moreover, the hepatitis B related antigen has been found in the serum of eight of 138 chimpanzees tested. The antigen has persisted for at least 5 years in three of these animals. This antigen has not been found in the serum of 99 baboons and 11 gibbons. Antigenemic chimpanzees were found to have histologic evidence of chronic persisting hepatitis. HB Ab was found in six of 138 chimpanzees and was transient in three of these animals. The chimpanzee antigen carrier provides a useful animal model for study of the hepatitis B virus carrier state and approaches to its therapy.
It appears likely that chronic HB Ag carriers are also hepatitis B virus carriers since blood containing the antigen has given rise to hepatitis in at least five out of eight recipients in one study and nine out of 12 in a second.
The presence of HB Ag and HB Ab may be quantitatively detected by agar gel diffusion using the methodology described by Prince, Proc. Nat'l. Acad. Sci. U.S.A. 60:814-821 (1968), by IEOP as described by Prince and Burke, Science 169:593-595 (1970), by passive hemagglutination (HA) and hemagglutination inhibition (HAI) as described by Vyas and Shulman, Science 170:332-333 (1970) and more recently by the direct radio-immuno assay (RIA) of Ling and Overby utilizing Ausria kits supplied by Abbott Laboratories.
It has been estimated that the plasma of hepatitis B virus carriers contains about 0.1 to 1.0 mg of antigen associated protein per ml of plasma. Carrier plasma may therefore serve as a useful source of antigen for production of vaccines.
Prince, in his U.S. patent application Ser. No. 301,347, filed Oct. 27, 1972, the entirety of which is hereby specifically incorporated by reference, discloses a vaccine against type B hepatitis infections, a method for production of such a vaccine and a vaccination process which makes use of the vaccine. Specifically, Prince's vaccine includes HB Ag particles having a diameter of from about 16 to about 30 nanometers in a physiologically acceptable carrier. The vaccine is substantially free of the probably infectious HB Ag particles having a diameter of about 40 to 45 nm.
Prince, in his above identified application, makes use of a purification process involving Freon extraction and methanol precipitation followed by zonal ultracentrifugation. The zonal ultracentrifugation procedures disclosed by Bond and Hall, J. Infect. Dis. 125:263-268 (1972) are disclosed by Prince as being appropriate for separation and purification of HB Ag into its morphologic forms; however, the procedures disclosed are hazardous and therefore special safety facilities are required. Moreover, these zonal ultracentrifugation procedures are extremely costly and the large total quantity of material which is fed to the ultracentrifuge in the method of application Ser. No. 301,347 per unit of product results in an extremely high cost of production for the purified antigen. Accordingly, less expensive methods for production of HB Ag are desirable so that the vaccines produced therefrom will be generally available to the public at a price which can be borne without substantial hardship on the part of either the individual or the government.
Polson, in U.S. Pat. No. 3,415,804, discloses methods for fractionating mixtures of proteinacious substances using polyethylene glycol (PEG) as a dispersibility depressant to produce a liquid phase containing one fraction in dispersion and a solid phase containing a second fraction. Utilizing the Polson method, which involves the use of a pH of 7.0, a temperature of 21.degree.C and protein concentrations less than 0.4 grams per 100 ml, a polyethylene glycol concentration greater than 12% is required to precipitate .alpha.-globulins and albumin. Polson suggests that the higher the protein concentration, the greater the overlap between fractions. Moreover, although HB Ag is normally considered to be associated with the .alpha.-globulin fractions, Polson does not disclose any method suitable for separating HB Ag from any of the fractions obtained from plasma. Further, very large concentrations of PEG are required by Polson to effect his desired fractionations.
De Rizzo, Pandey, Wallis and Melnik, Inf. and Imm. 6:335-338 (1972) have disclosed a two step method for concentrating and purifying HB Ag utilizing PEG and polyelectrolyte 60 (a cross-linked copolymer of isobutylene maleic anhydride). In the first step, plasma having a protein concentration of 79.9 mg/ml is subjected to PEG precipitation at a pH of 4.6 and a temperature of 25.degree.C, utilizing a PEG concentration of 8%. It is indicated that the precipitate contains 100% of the original HB Ag and only 12.5% of the original proteins. The proteins in the precipitate comprise 2 mg of albumin per ml, 3 mg of .alpha..sub.2 -globulin per ml and 5 mg of gamma-globulin per ml. It was reported tht all of the .alpha..sub.1, .beta., and .beta..sub.2 globulins remained in the liquid phase. The precipitate thus obtained, by precipitation with PEG, was then subjected to concentration and purification of the HB Ag therein utilizing polyelectrolyte 60. Eight-fold purification of the HB Ag in the PEG precipitation step was reported by De Rizzo et al.; however, attempts to utilize the De Rizzo et al. method by the present applicants have indicated that the procedure provides only a two-fold purification. Furthermore, it has been found that the De Rizzo et al. method is not reproducible as described.
Blumberg and Millman, in U.S. Pat. No. 3,636,191, have disclosed methods for producing a vaccine against viral hepatitis wherein plasma containing the antigen is subjected to ultracentrifugation, enzyme digestion, column gel filtration, differential density centrifugation in a solution of sucrose, dialysis, differential density centrifugation in a solution of cesium chloride and dialysis. The disclosed method is very expensive and is not suitable for the large scale purification of HB Ag. Further, it has been found that the antigen may be altered by proteolytic digestion. Moreover, the procedure disclosed does not appear to separate the larger HB Ag associated particles which occur in donor carrier plasma.